Citations

Peter J. Psaltis, 2011 Finalist and Presenting Author

Background: Hematopoietic stem cells and the vasculature share a unique relationship during development, in the adult bone marrow and in the setting of extramedullary hematopoiesis within diseased calcified vessels. We hypothesized that the adult vasculature may contain progenitor cells with the capacity to reconstitute multi-lineage hematopoietic cells. Methods: Aortas from normal and ApoE −/− adult mice were disaggregated with collagenase and elastase. Disaggregates were cultured in methylcellulose for quantification of short and long term hematopoietic colony forming units (CFU). Flow cytometry assessed various populations of hematopoietic progenitor cells. Aortic disaggregates (or bone marrow or blood) from ROSA26 and GFP mice were transplanted into sublethally irradiated C57BL/6 recipients (n=6) via tail vein injections. Bone marrow cells from the primary recipients were transplanted at 4 months to secondary C57BL/6 recipients (n=6). Mice were sacrificed 3 months later. Blood and bone marrow were analyzed. Results: In short term culture in methylcellulose, aortic disaggregates generated erythroid, macrophage, granulocyte and megakaryocyte CFU, with predominance of macrophage colonies, primarily from adventitia (p<0.05) and a greater frequency was found in Apo E −/− mice (p<0.05). Long term culture defined that the combined frequency of the hematopoietic precursors approximates the frequency found in bone marrow (1:47,277). Flow cytometry of the aortic disaggregates indicates that among lin-/c-kit+/sca-1+ cells a small population of hematopoietic stem cells (CD150+endoglin+) and a population of multipotent progenitor cells (CD150− endoglin−) exist. Infusion of aortic disaggregates generated bone marrow, blood and spleen chimerism in primary and secondary transplanted mice at 3– 4 months. Flow cytometry of peripheral cells demonstrated multilineage hematopoietic cells. Conclusions: For the first time, we have identified a variety of progenitor cell populations in the aorta capable of hematopoiesis. Infusion of these cells into sublethally irradiated mice results in stable chimeras. These data support the presence of novel aortic hematopoietic progenitor cells.

Abstract Abstract 4319 C-type natriuretic peptide (CNP) is known to exert pleiotropic effects on a variety of cell types through binding to its receptor, natriuretic peptide receptor type B (NPR-B) and activation of the cytoplasmic intracellular guanylyl cyclase domain. Although nitric oxide-mediated activation of cyclic guanosine monophosphate (cGMP) is known to induce megakaryocyte apoptosis and regulate platelet activation, the effects of CNP on these cells is yet to be defined. In this study for the first time we have addressed the potential role of CNP and NPR-B in megakaryocyte and platelet biology. We identified NPR-B on murine megakaryocytes by immunostaining demonstrating co-localization of CD41 and NPR-B. CNP induced cGMP production in megakaryocytes compared to a mutant form of CNP containing a single amino acid substitution (Lbab) (67.24 ± 10.65 fmol/well vs. 17.18 ± 2.089 fmol/well; P<0.05). Subsequently, CNP was shown to reduce megakaryocyte colony formation compared to Lbab and this effect was dose-dependent: Lbab 500nM 26.75 ± 5.618 CFU-M vs. 15.25 ± 2.750 CFU-M for CNP 500nM and Lbab 1000 nM 27.50 ± 2.102 CFU-M vs. 12.25 ± 1.493 CFU-M for CNP 1000nM; P<0.05). Furthermore, CNP also resulted in smaller-sized colonies, with altered cell morphology. To verify these findings, wild-type C57/BL6 mice were treated with daily injections of either saline, 1uM CNP or 1uM Lbab for one week, following which megakaryocytes were isolated and cultured. Colony formation was diminished in mice injected with CNP compared to mice receiving saline or Lbab peptide (125.3 ± 18.54 CFU-M for saline; 107.0 CFU-M ± 20.04 for Lbab and 20.00 ± 3.674 CFU-M for CNP; P<0.05). In addition platelet numbers were measured in the aforementioned mice and the CNP treated mice had significantly lower platelet counts compared to the mice treated with Lbab (832.0 ± 13.19 ×103/ul for CNP vs. 778.0 ± 12.00 ×103/ul for Lbab; P<0.05). Taken together, these data indicate that CNP may play a role in the development, maturation and/or survival of megakaryocytes. Using flow cytometry we identified that 47% of CD41+ murine platelets co-express NPR-B. In addition, this expression of NPR-B was confirmed by immunocytostaining and western blotting in both human and murine platelets. Interestingly, despite the expression of NPR-B, and unlike megakaryocytes, CNP (similar to the mutant peptide) did not increase cGMP production in platelets (1.652 ± 0.016 fmol/well for CNP and 1.606 ± 0.01550 fmol/well for Lbab). Further, although preliminary results did not indicate that CNP alters platelet secretion or aggregation, exposure to the peptide profoundly inhibited adhesion of platelets to collagen (53 ± 5 adhered platelets with Lbab, 32 ± 3 with CNP, relative reduction 42%; P<0.05). In summary, these results provide for the first time evidence that the NPR-B receptor is expressed on both megakaryocytes and platelets. Our studies suggest that CNP might have both cGMP dependent and independent functions in megakaryocytes and platelets and further studies will assist in delineating the mechanisms by which CNP exerts its actions on these cells. Disclosures: No relevant conflicts of interest to declare.

Human islet amyloid polypeptide (IAPP) is a 37-residue peptide that is co-secreted with insulin by the β-cell and might be involved in the pathogenesis of non-insulin-dependent diabetes mellitus. We developed an improved assay in vitrobased on the fluorescence of bound thioflavin T to study factors affecting amyloidogenesis. Monomeric IAPP formed amyloid fibrils, as detected by increased fluorescence and by electron microscopy. Fluorimetric analysis revealed that the initial rate of amyloid formation was: (1) proportional to the peptide monomer concentration, (2) maximal at pH 9.5, (3) maximal at 200 mM KCl, and (4) proportional to temperature from 4 to 37 °C. We found that 5-fold and 10-fold molar excesses of proinsulin inhibited fibril formation by 39% and 59% respectively. Insulin was somewhat more potent with 5-fold and 10-fold molar excesses inhibiting fibril formation by 69% and 73% respectively, whereas C-peptide had no effect at these concentrations. Thus at physiological ratios of IAPP to insulin, insulin and proinsulin, but not C-peptide, can retard amyloidogenesis. Because insulin resistance or hyperglycaemia increase the IAPP-to-insulin ratio, increased intracellular IAPP compared with insulin expression in genetically predisposed individuals might contribute to intracellular amyloid formation, β-cell death and the genesis of non-insulin-dependent diabetes mellitus.

Rationale : Macrophages regulate blood vessel structure and function in health and disease. The origins of tissue macrophages are diverse, with evidence for local production and circulatory renewal. Objective : We identified a vascular adventitial population containing macrophage progenitor cells and investigated their origins and fate. Methods and Results : Single-cell disaggregates from adult C57BL/6 mice were prepared from different tissues and tested for their capacity to form hematopoietic colony-forming units. Aorta showed a unique predilection for generating macrophage colony-forming units. Aortic macrophage colony-forming unit progenitors coexpressed stem cell antigen-1 and CD45 and were adventitially located, where they were the predominant source of proliferating cells in the aortic wall. Aortic Sca-1 + CD45 + cells were transcriptionally and phenotypically distinct from neighboring cells lacking stem cell antigen-1 or CD45 and contained a proliferative (Ki67 + ) Lin − c-Kit + CD135 − CD115 + CX 3 CR1 + Ly6C + CD11b − subpopulation, consistent with the immunophenotypic profile of macrophage progenitors. Adoptive transfer studies revealed that Sca-1 + CD45 + adventitial macrophage progenitor cells were not replenished via the circulation from bone marrow or spleen, nor was their prevalence diminished by depletion of monocytes or macrophages by liposomal clodronate treatment or genetic deficiency of macrophage colony-stimulating factor. Rather adventitial macrophage progenitor cells were upregulated in hyperlipidemic ApoE −/− and LDL-R −/− mice, with adventitial transfer experiments demonstrating their durable contribution to macrophage progeny particularly in the adventitia, and to a lesser extent the atheroma, of atherosclerotic carotid arteries. Conclusions : The discovery and characterization of resident vascular adventitial macrophage progenitor cells provides new insight into adventitial biology and its participation in atherosclerosis and provokes consideration of the broader existence of local macrophage progenitors in other tissues.

Abstract —Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours ( P <0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively ( P <0.05), and these growth factors accounted for ≈50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.

Background —Plaque disruption and exposure of subendothelial procoagulants such as tissue factor (TF) to circulating factor VII/VIIa (FVII/VIIa) lead to intravascular thrombosis. Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of TF-induced coagulation that binds to factor Xa and the TF-FVIIa catalytic complex in a two-step process. The aim of this study was to determine the expression of TFPI within human atherosclerotic plaque and its role in modulation of TF activity. Methods and Results —We measured the level of TFPI antigen in human carotid plaque and determined the relationship between TFPI and TF activity within plaque. Furthermore, we examined the biological activity and immunolocalization patterns of TFPI within carotid plaque. TFPI was detectable (TFPI+ group) in 22 of 34 specimens (mean±SEM, 404.4±91.8 pg/mg) and undetectable (TFPI− group) in 12 of 34 specimens. In the TFPI− group, normalized TF activity was significantly greater than that in the TFPI+ group (0.28±0.04 vs 0.14±0.02 U/pg, P =0.002). Furthermore, neutralization of TFPI activity using a polyclonal antibody resulted in an 8-fold increase in TF activity in the TFPI+ group ( P =0.001) but had no effect in the TFPI− group. Immunostaining for TFPI showed localization to endothelial cells, vascular smooth muscle cells within the fibrous cap region of the plaque, and macrophages within the shoulder region of the plaque. Conclusions —Taken together, these data suggest that biologically active TFPI is present within human atherosclerotic plaque and is associated with attenuated TF activity.

Objective— Tissue factor pathway inhibitor (TFPI) is the primary regulator of the tissue factor (TF) coagulation pathway. As such, TFPI may regulate the proangiogenic effects of TF. TFPI may also affect angiogenesis independently of TF, through sequences within its polybasic carboxyl terminus (TFPI C terminus [TFPIct]). We aimed to determine the effects of TFPI on angiogenesis and the role of TFPIct. Methods and Results— Transgenic overexpression of TFPI attenuated angiogenesis in the murine hindlimb ischemia model and an aortic sprout assay. In vitro, TFPI inhibited endothelial cell migration. Peptides within the human TFPIct inhibited endothelial cell cord formation and migration in response to vascular endothelial growth factor (VEGF) 165 but not VEGF121. Furthermore, exposure to human TFPIct inhibited the phosphorylation of VEGF receptor 2 at residue Lys951, a residue known to be critical for endothelial cell migration. Finally, systemic delivery of a murine TFPIct peptide inhibited angiogenesis in the hindlimb model. Conclusion— These data demonstrate an inhibitory role for TFPI in angiogenesis that is, in part, mediated through peptides within its carboxyl terminus. In addition to its known role as a TF antagonist, TFPI, via its carboxyl terminus, may regulate angiogenesis by directly blocking VEGF receptor 2 activation and attenuating the migratory capacity of endothelial cells.

Background— Hematopoiesis originates from the dorsal aorta during embryogenesis. Although adult blood vessels harbor progenitor populations for endothelial and smooth muscle cells, it is not known if they contain hematopoietic progenitor or stem cells. Here, we hypothesized that the arterial wall is a source of hematopoietic progenitor and stem cells in postnatal life. Methods and Results— Single-cell aortic disaggregates were prepared from adult chow-fed C57BL/6 and apolipoprotein E–null (ApoE −/− ) mice. In short- and long-term methylcellulose-based culture, aortic cells generated a broad spectrum of multipotent and lineage-specific hematopoietic colony-forming units, with a preponderance of macrophage colony-forming units. This clonogenicity was higher in lesion-free ApoE −/− mice and localized primarily to stem cell antigen-1–positive cells in the adventitia. Expression of stem cell antigen-1 in the aorta colocalized with canonical hematopoietic stem cell markers, as well as CD45 and mature leukocyte antigens. Adoptive transfer of labeled aortic cells from green fluorescent protein transgenic donors to irradiated C57BL/6 recipients confirmed the content of rare hematopoietic stem cells (1 per 4 000 000 cells) capable of self-renewal and durable, low-level reconstitution of leukocytes. Moreover, the predominance of long-term macrophage precursors was evident by late recovery of green fluorescent protein–positive colonies from recipient bone marrow and spleen that were exclusively macrophage colony-forming units. Although trafficking from bone marrow was shown to replenish some of the hematopoietic potential of the aorta after irradiation, the majority of macrophage precursors appeared to arise locally, suggesting long-term residence in the vessel wall. Conclusions— The postnatal murine aorta contains rare multipotent hematopoietic progenitor/stem cells and is selectively enriched with stem cell antigen-1–positive monocyte/macrophage precursors. These populations may represent novel, local vascular sources of inflammatory cells.

Abstract Pulmonary hypertension (PH) is a commonly recognized complication of chronic respiratory disease. Enhanced vasoconstriction, pulmonary vascular remodeling, and in situ thrombosis contribute to the increased pulmonary vascular resistance observed in PH associated with hypoxic lung disease. The tissue factor pathway regulates fibrin deposition in response to acute and chronic vascular injury. We hypothesized that inhibition of the tissue factor pathway would result in attenuation of pathophysiologic parameters typically associated with hypoxia-induced PH. We tested this hypothesis using a chronic hypoxia–induced murine model of PH using mice that overexpress tissue factor pathway inhibitor (TFPI) via the smooth muscle–specific promoter SM22 (TFPISM22). TFPISM22 mice have increased pulmonary TFPI expression compared with wild-type (WT) mice. In WT mice, exposure to chronic hypoxia (28 d at 10% O2) resulted in increased systolic right ventricular and mean pulmonary arterial pressures, changes that were significantly reduced in TFPISM22 mice. Chronic hypoxia also resulted in significant pulmonary vascular muscularization in WT mice, which was significantly reduced in TFPISM22 mice. Given the pleiotropic effects of TFPI, autocrine and paracrine mechanisms for these hemodynamic effects were considered. TFPISM22 mice had less pulmonary fibrin deposition than WT mice at 3 days after exposure to hypoxia, which is consistent with the antithrombotic effects of TFPI. Additionally, TFPISM22 mice had a significant reduction in the number of proliferating (proliferating cell nuclear antigen positive) pulmonary vascular smooth muscle cells compared with WT mice, which is consistent with in vitro findings. These findings demonstrate that overexpression of TFPI results in improved hemodynamic performance and reduced pulmonary vascular remodeling in a murine model of hypoxia-induced PH. This improvement is in part due to the autocrine and paracrine effects of TFPI overexpression.